Bacteria exhibit an amazing diversity of shapes and sizes that are important for their survival in the environment. For example, some pathogens adopt specific morphologies that facilitate infection. Filamentation of uropathogenic E. coli subverts innate defenses during the infection process, and the helical shape of Helicobacter pylori is important for colonization of the stomach. In bacteria, morphologies and growth modes are interconnected. Recent studies have shown that bacterial growth involves spatially structured, zonal modes of peptidoglycan (PG) cell wall synthesis. The general goal of this research is to understand how bacteria achieve changes in morphology and modes of growth. Constraining PG synthesis to specific zones can generate alternate modes of growth and reproduction, or generate different morphologies. This study focuses on a protein, SpmX, which controls the synthesis of thin cylindrical extensions of the cell envelope layers, called stalks, which provide a powerful non-essential model of zonal PG synthesis. Investigating the structure and function of SpmX will increase our understanding of the principles that govern the spatiotemporal regulation of PG synthesis, will reveal the basic principles that control zonal growth, and add to the knowledge base required to direct efforts towards impeding their persistence, proliferation, and pathogenicity. The project has four major aims. 1. The structure of SpmX will be analyzed, using X-ray crystallography and NMR, to provide a molecular basis for the structure-function analysis of the different domains of SpmX. 2. Mutational and biochemical analysis of SpmX activity will be performed. In order to understand how SpmX directs zonal PG synthesis, the function of key catalytic, PG binding, protein-protein interaction, and oligomerization residues will be tested by site-directed mutagenesis, and the effect of these mutations will be studied by a variety of in vivo and in vitro methods. 3. The mechanism of SpmX localization and its interacting partners involved in zonal PG synthesis will be identified by combining high-throughput genetic screens with proteomic and candidate approaches. The function of two newly identified stalk synthesis proteins, the bactofilin BacA and the putative endopeptidase LytM will be studied, and the model, based on a recent discovery, that polar PG synthesis plays an important role in the localization of SpmX will be tested. 4. Building on compelling evidence that indicates that the SpmX muramidase domain occupies both the cytoplasm and the periplasm, SpmX topology and its importance in localization and regulation will be studied. Topological switching could provide spatiotemporal control over zonal PG activity. This work will provide a detailed understanding of zonal growth in a tractable, nonessential bacterial organelle. The general principles uncovered should apply to other cases of zonal PG synthesis that direct bacterial growth and division, and these principles can be used to improve the development of new antibacterial strategies. Moreover, the findings may provide direct mechanistic insight into zonal PG synthesis of pathogens such as Brucella and Rickettsia.